Erythrocyte-derived depressing factor (EDDF) shows significant protective effects on blood vessels from hypertensive rats, by regulating vascular reactivity, calcium homeostasis, DNA synthesis, and cell cycle progression in vascular smooth muscles (VSMCs). Arteries from hypertensive and aging people have high levels of accumulated calcium. However, in the life span of experimental animals commonly used, arterial calcium content does not reach cytotoxic levels observed in human. An overdose of vitamin D(3) results in a rapid arterial calcium overload. Using rats with arterial calcinosis and age- and gender-matched Wistar controls, we investigated whether EDDF has beneficial effect on blood vessels from animals with arterial calcinosis. Blood vessel functions were impaired in rats with arterial calcinosis, as indicated by decreased Ca(2+)-ATPase activity, increased vasoconstrictor responses to alpha1 adrenoceptor agonist phenylephrine and increased ERK1/2 phosphorylation. Arterial calcium overload also impaired the morphological integrity of VSMCs. EDDF restored the abovementioned abnormalities caused by arterial calcinosis, and inhibited cell cycle progression of VSMCs induced by angiotensin II. In conclusion, EDDF may protect blood vessels from animals with arterial calcinosis, which is mediated by regulating calcium homeostasis, vascular reactivity and cell cycle progression as well as by improving morphological integrity of VSMCs.